Proteins are complex, three-dimensional substances comprising one or more long, folded polypeptide chains. These chains, in turn, consist of small chemical units called amino acids. All amino acids contain carbon, hydrogen, oxygen, and nitrogen. Some also contain sulfur. A "peptide" is a compound that includes two or more amino acids. The amino acids link together in a line to form a peptide chain. There are 20 different naturally occurring amino acids involved in the biological production of peptides, and any number of them may be linked in any order to form a peptide chain. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, utilizing L-amino acids, D-amino acids, or various combinations of amino acids of the two different configurations. Some peptide chains contain only a few amino acid units. Short peptide chains, e.g., having less than ten amino acid units, are sometimes referred to as "oligopeptides", where the prefix "oligo" signifies "few". Other peptide chains contain a large number of amino acid units, e.g., up to 100 or more, and are referred to a "polypeptides", where the prefix "poly" signifies "many". Still other peptide chains, containing a fixed number of amino acid units are referred to using a prefix that signifies the fixed number of units in the chain, e.g., an octapeptide, where the prefix "octa" signifies eight. (By convention, a "polypeptide" may be considered as any peptide chain containing three or more amino acids, whereas an "oligopeptide" is usually considered as a particular type of "short" polypeptide chain. Thus, as used herein, it is understood that any reference to a "polypeptide" also includes an oligopeptide. Further, any reference to a "peptide" includes polypeptides, oligopeptides, and the like.) Each different arrangement of amino acids forms a different polypeptide chain. The number of chains--and hence the number of different proteins--that can be formed is practically unlimited.
A drug is a chemical substance administered to a living organism with the intention of bringing about some desired result, such as preventing or curing disease. The desired result is usually achieved through an appropriate physical or chemical interaction between the administered drug and compounds found in living tissue.
All living things contain proteins. The structures of a cell are built of proteins. Some proteins, known as enzymes, speed up the chemical reactions of life. They help digest food, help produce energy, and assist in building other proteins. A single cell may contain many hundreds of enzymes. Other peptides and proteins, known as hormones, regulate chemical activities throughout the body. Still other proteins are antibodies that recognize and attach foreign bodies.
Drug design has historically involved "discovering" a particular chemical substance that interacts in some way with receptors, e.g., proteins in the living cells of a mammalian body. As proteins are made up of polypeptides, it is not surprising that some effective drugs are also peptides, or are patterned after peptides. Generally, for two peptides to effectively interact with each other, e.g., one as a protein receptor and the other as a drug, it is necessary that the complex three-dimensional shape ("conformation") of one peptide assume a compatible conformation that allows the two peptides to fit and bind together in a way that produces a desired result. In such instance, the complex shape or conformation of a first peptide has been compared to a "lock" and the corresponding requisite shape or conformation of the receptor as a "key" that unlocks (i.e., produces the desired result within) the first peptide. This "lock-and-key" analogy emphasizes that only a properly conformed key (second peptide or compound patterned thereafter) is able to fit within the lock (first peptide) in order to "unlock" it (produce a desired result). Further, even if the key fits in the lock, it must have the proper composition in order for it to perform its function. That is, the second peptide must contain the right elements in the right spatial arrangement and position in order to properly bind with the first peptide, e.g., receptor protein. Discovering or predicting the proper conformation or shape of the key, or second peptide or compound patterned thereafter, is thus a major objective of any drug design.
To better understand and appreciate the obstacles involved in discovering or predicting the conformation of an oligopeptide or polypeptide, reference is made to FIG. 1 where the conventional chemical representation of a neutral oligopeptide, consisting of four amino acids, is shown. Depending on the pH of the medium in which the peptide is present, the peptide can contain a variety of charged species, i.e., one or more ammonium species, carboxyl anions, etc. Note that the molecule represented in FIG. 1 has a terminal amino (NH.sub.2) group at the left end of the chain, as oriented in the figure, and a terminal free carboxyl (--COOH) group at the right end of the chain. These ends of the polypeptide are called the amino (NH.sub.2) terminal end and the carboxyl (--COOH) terminal end, respectively. This same terminology applies in the case of proteins. By convention, the NH.sub.2 -terminal amino acid in an oligopeptide of the polypeptide chain of a protein is called the first amino acid or the first "residue". The next amino acid in the chain is called the second amino acid or the second residue, and so on, throughout the length of the chain.
The R groups shown in FIG. 1, i.e., R.sup.1, R.sup.2, R.sup.3, and R.sup.4, symbolize various "pendant groups" of the chain The pendant groups are always attached to the alpha carbon (C.sup..alpha.) atom (i.e., to the carbon-hydrogen (CH) component of the chain, as shown in FIG. 1). A pendant group may comprise a simple or complex group or moiety, having physical dimensions that can vary significantly compared to the dimensions of the chain.
There are a number of factors that play important roles in determining the total structure of a protein or polypeptide. First, the peptide bond, i.e., that bond which joins the amino acids in the chain together, is a covalent bond. This bond is planar in structure, being essentially a substituted amide. An "amide" is any of a group of organic compounds containing the radical: ##STR1## The planar peptide bond may be represented as depicted in FIG. 2. Because the O.dbd.C and the C--N atoms lie in a relatively rigid plane, free rotation does not occur about these axes. Hence, a plane, schematically depicted in FIG. 2 by the dotted line 12, and sometimes referred to as an "amide plane" or "peptide plane", is formed wherein lie the oxygen (O), carbon (C), nitrogen (N), and hydrogen (H) atoms of a given amino acid or residue. At opposite corners of this amide plane are located the .alpha.-carbon (C.sup..alpha.) atoms. Since there is substantially no rotation about the rigid O.dbd.C and C--N atoms in the peptide or amide plane, a polypeptide chain thus comprises a series of planar peptide linkages joining the C.sup..alpha. atoms. The C.sup..alpha. atoms thus serve as swivel points or centers for a polypeptide chain as shown in FIG. 2B. In FIG. 2B, the shaded areas 12 represent the peptide or amide planes. Note that each plane is coupled to the adjacent plane through a C.sup..alpha. atom.
A second factor that plays an important role in defining the total structure or conformation of a polypeptide or protein is the angle of rotation of each amide or peptide plane about the common C.sup..alpha. linkage. Advantageously, assuming that the O, C, N and H atoms remain in the amide plane (which is usually a valid assumption, although there may be some slight deviations from planarity of these atoms for some conformations), these angles of rotation completely define the polypeptide's structure, at least the structure as it exists between adjacent residues. These angles of rotation are illustrated in FIG. 2C. In FIG. 2C, two amide planes are shown, represented by the dotted lines 12' and 12". These two planes are joined by a common C.sup..alpha. atom 13 that is the corner of each plane. The angle of rotation of the plane 12' relative to the common C.sup..alpha. -atom 13 is defined as .phi.. The angle of rotation of the plane 12" relative to the C.sup..alpha. atom 13 is defined as .psi.. The two angles .phi., .psi. thus substantially define the peptide structure for the main chain of a particular residue of the peptide chain. A set of the angles .phi..sub.i, .psi..sub.yi where the subscript i represents a particular residue of a polypeptide chain, thus effectively defines the total polypeptide secondary structure.
It is noted that the conventions used in defining the .phi., .psi. angles, i.e., the reference points at which the amide planes form a zero degree angle, and the definition of which angle is .phi., and which angle is .psi., for a given polypeptide, are defined in the literature. See, e.g., Ramachandran et al., "Conformation of Polypeptides," Adv. Prot. Chem. 23, 283-437 (1968), at pages 285-94, which pages are incorporated herein by reference.
Thus, a polypeptide structure bends, folds or flexes at each C.sup..alpha. swivel point. In a particular environment, and depending upon the particular side chains that may be attached to the polypeptide, some of these bends or folds may be stable, i.e., the .phi., .psi. angles will not change. In many environments, however, the .phi., .psi. angles will not be stable, and the polypeptide chain will dynamically fold and bend (much as a snake swimming in water) as subjected to external or internal forces. Such forces may originate from numerous sources, such as ions, or molecules in the medium wherein the polypeptide is located (external forces) that either attract or repel a given atom or group of atoms within the polypeptide. Often, however, these forces originate from within the polypeptide itself, or within one of its pendant groups, as the chain folds back on itself and one residue or pendant group of the polypeptide comes in close proximity to another residue or pendant group chain of the polypeptide.
To illustrate the manner in which a polypeptide chain may bend or fold, FIG. 3A conceptually shows a polypeptide that has assumed a helical conformation. The helix shown in FIG. 3A is a specific configuration called the right-handed .alpha. helix. This structure, exhibiting 3.6 amino acid residues per turn, is representative of numerous known stable peptide structures. Stability results due to hydrogen bonding between an --NH-- group in the helix and the --C.dbd.O group of the fourth amino acid down the chain. Under the conditions shown in FIG. 3A, the .phi. value is about -60.degree., and the .psi. value is about -40.degree..
Since the C.sup..alpha. atom is the swivel point for the chain, the R groups (side or pendant groups) associated with the C.sup..alpha. atom become extremely important in defining the ultimate peptide conformation.
In general, just as a flexible rope can assume an infinite number of shapes, including highly symmetrical shapes, such as a helix, or asymmetrical shapes involving all kinds of contortions, a polypeptide chain can conceptually also assume an infinite number of shapes. Many of the possible shapes, however, are unstable, because the internal and external molecular attraction and/or repulsion forces will not permit such shapes to persist. These forces act to move or change the polypeptide conformation away from unstable conformations toward a stable conformation. A stable conformation is one where the internal and external molecular attraction and/or repulsion forces fail to destabilize or push the existing conformation toward another conformation.
Most polypeptide structures exhibit several conformations that are stable, some more so than others. The most stable conformations are the most probable. A conformation may change from one stable conformation to another through the application of sufficient energy to cause the change. Given the opportunity to freely move, fold and/or bend, a given polypeptide chain will eventually assume a stable conformation. The most probable conformation that is assumed is the one that would take the most energy to undo. This most probable conformation is referred to herein as the "global minimum". Other stable conformations are less probable, but may readily be assumed, and are referred to herein as a "local minimum" or "local minima". A conformation that represents a local minimum could thus be changed, through application of an external force, to another stable conformation which is either another, different local minimum or the global minimum. Being able to distinguish whether a given conformation represents a local minimum or the global (or most probable) minimum remains a significant problem when peptide simulation is performed.
Shown in FIG. 3B is a complex three-dimensional conformation of a polypeptide, typical of many proteins, stabilized by noncovalent bonds. Shown in FIG. 3C are two such complex polypeptide conformations, closely packed with each other. FIG. 3C is thus illustrative of the "lock-and-key" analogy associated with drug design. Only by designing the conformation of one peptide to allow it to fit within the conformation of the other peptide and bind thereto will the desired interaction between the two peptides take place. Rational drug design thus includes not only knowing or predicting the conformation of a desired protein receptor peptide, but also being able to control and predict the conformation of a drug peptide that is to interact with the receptor peptide.
At this juncture, it will be helpful to define some common terms used to define the complex structure of proteins and polypeptides. A primary structure is one wherein the number and precise sequence of amino acids in the polypeptide is known. The peptide linkage between each of the amino acid residues is implied, but no other forces or bonds are indicated by use of the term "primary structure". Thus, the chemical representation of the peptide shown in FIG. 1 defines its primary structure. A secondary structure refers to the extent to which a polypeptide chain possesses any helical or other stable structure, such as shown in FIG. 3A. A secondary structure will thus have a set of angles, .phi..sub.i, .psi..sub.i for each residue i of the chain. A tertiary structure is a term used to refer to the tendency for the polypeptide to undergo extensive coiling or folding to produce a complex, somewhat rigid three-dimensional structure, such as is shown in FIG. 3B. A quaternary structure is a term used to define the degree of association between two or more polypeptides, e.g., between two tertiary structures, such as a target peptide and a receptor, as suggested by FIG. 3C.
To the four basic structures defined above, some authors have further described and coined terms for intermediate structures, e.g., supersecondary and domain structures. Whereas a secondary structure is used to refer to the regular arrangements of the polypeptide backbone, a "supersecondary" structure is used to define aggregates of the secondary structure. "Domain" structures are used to refer to well-separated parts within globular proteins, i.e., within tertiary structures. See, e.g., Linderstrom-Lang, et al., "Protein Structure and Enzyme Activity, " The Enzymes, (P. D. Boyer, Ed.), 1:443-510, Academic Press, New York (1959); and Schulz et al., Principles of Protein Structure, Springer-Verlag, New York (1984).
Those skilled in the art will recognize that the above description of a polypeptide chain and the factors that define its total structure are somewhat simplified. However, the above description nonetheless provides a sufficient background for understanding the present invention. For a more thorough description of polypeptide structure, see, e.g., Ramachandran et al., "Conformation of Polypeptides," Adv. Prot Chem. 23, 283-437 (1968).
With the foregoing as background, it is thus seen that drug design involving polypeptides requires identifying and defining a first peptide with which the designed drug is to interact, and using the first target peptide to define the requirements for a second peptide. With such requirements defined, the goal is then to find or prepare an appropriate peptide or non-peptide ligand that meets all or substantially all of the defined requirements which can hopefully be used as the administered drug.
As a practical matter, however, this process of drug design has proven to be very difficult. In the first place, many of the protein peptides with which the administered drug is to interact do not themselves exhibit stable conformations, so it is difficult to use such protein peptides in trying to set the requirements for a drug peptide. While a particular application, e.g., a particular protein peptide, may provide some clues as to an appropriate primary or secondary structure that an administered drug peptide might assume, it may provide few clues as to the best conformation for the drug peptide. Further, even if a desired conformation of a compound of interest were identifiable, being able to administer such compound to a patient in a form which maintains such conformation may not be possible. That is, for a given application, a preferred conformation of the compound of interest may not be sufficiently stable to impart the desired effect to the recipient organism.
In view of the above difficulties, the best that has been achieved to date in rational drug design is to search for an appropriate compound that could be administered as a drug and that provides a stable secondary or tertiary structure. Once identified, this compound is tested to see if it is bioactive (i.e., to see if it has the capacity to interact with a desired receptor peptide). If so, it is further tested to see if the desired beneficial results are achieved.
Thus, much of the drug design heretofore conducted has involved intensive efforts aimed at searching for bioactive peptides and testing any that are so identified. It is thus evident that what is needed is a method or technique of predicting the best conformation for a peptide drug; and, once found, providing a means for maintaining this conformation so that it can be further tested, e.g., for bioactivity.
The process of drug design is further complicated by the metabolic degradation of the amide bonds of many polypeptide chains. That is, even assuming a given peptide drug having a desired conformation is identified, and further assuming that this desired conformation can be maintained, the actual peptide bonds linking the amino acid residues in the peptide chain may break apart when the peptide drug is orally administered. Once such bonds are broken, all that is left are portions (moieties) of the polypeptide chain which do not provide the needed conformation for the peptide drug to perform its intended task (i.e., the "key" has broken apart, and a broken key is not able to unlock the lock). Hence, a method or technique is needed for preventing the amide bonds of a peptide drug from breaking down, prior to the realization of the desired effect, upon administration of the peptide drug. In other words, it is desired that the peptide drug survive in its active form until it reaches the site where it exerts its biological effect.
Several techniques are known in the art in an attempt to address the above problems. For example, it is known in the drug design art to look for a substitute compound that mimics the conformation and desirable features of a particular peptide, e.g., an oligopeptide, once such peptide has been found, but that avoids the undesirable features, e.g., flexibility (loss of conformation) and bond breakdown. Such a compound that mimics a peptide is known as a "peptidomimetic". For example, morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin. However, to date, only limited success has been reported in these attempts, largely because it has been so difficult to identify the desired starting point, i.e., the conformation of the particular oligopeptide or other peptide that is to be mimicked. See, e.g., Spatola, A. F. Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (1983), which describes the use of the methylenethio bioisostere [CH.sub.2 S] as an amide replacement in enkephalin analogues; and Szelke et al., In peptides: Structure and Function, Proceedings of the Eighth American Peptide Symposium, (Hruby and Rich, Eds.); pp. 579-582, Pierce Chemical Co., Rockford, Ill. (1983), which describes renin inhibitors having both the methyleneamino [CH.sub.2 NH] and hydroxyethylene [CHOHCH.sub.2 ] bioisosteres at the Leu-Val amide bond in the 6-13 octapeptide derived from angiotensinogen. Hence, what is needed is a rational approach for identifying the most probable starting point for the design of a bioactive peptidomimetic.
It is also known in the art to use computer simulation in an attempt to predict a stable conformation of a peptide. That is, because a peptide is a sequence of amino acid residues, each containing known atoms bonded together in known molecules having known bonding lengths, with known electrostatic properties associated with each atom, it is possible to simulate a peptide structure on the computer. However, the difficulty with such computer simulations to date has been the propensity of such simulations to identify only "local minimum" conformations of the subject peptide, since the most probable conformation of the peptide may fall outside some of the parameters assumed for purposes of carrying out the simulation calculations. In addition, there is typically an enormous amount of computer time required to systematically examine all possible conformational possibilities of the peptide, particularly when more than just a short peptide is being simulated.
In order to shorten the amount of computer time required in such simulations, it is known to specify a starting point, e.g., a good estimate of the conformation of the stable peptide having a known amino acid sequence. Such estimate may be based on known data, e.g., as obtained using X-ray crystallography, or as predicted using model building of three-dimensional structures of homologous proteins when the three-dimensional structure of at least one of the proteins in the structure is known. Unfortunately, while such "starting points" do significantly shorten the amount of computer time required in such simulations, they also bias the final results. Frequently, such simulations end up identifying only a "local minimum" of the predicted peptide, with the most probable stable conformation of the peptide going undetected. What is clearly needed, therefore, is a method and technique of predicting the most probable stable peptide conformations using computer simulations that may be feasibly performed and that do not bias the final results.
The present invention advantageously addresses the above and other needs associated with drug design.